Original Article

Stratification of HPV-induced cervical pathology using the virally encoded molecular marker E4 in combination with p16 or MCM

  • Modern Pathology (2015) 28, 977993 (2015)
  • doi:10.1038/modpathol.2015.52
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High-risk human papillomavirus (HPV) types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16INK4a and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble nonproductive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated hematoxylin and eosin (H&E) image. Conventional grading of neoplasia was established by review panel, and compared directly with the composite molecular pathology visualized on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualized by standard H&E staining. Of the dual marker approaches, p16INK4a and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16INK4a was present. Extensive p16INK4a expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary molecular biomarker pairs such as MCM/E4 or, more promisingly, p16INK4a/E4 as an adjunct to conventional pathology.

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JD, HG, YS, and ZW were funded by the UK Medical Research Council. RvB was funded by the Stichting Pathologie Ontwikkeling en Onderzoek (SPOO) Foundation, The Netherlands.

Author information


  1. Department of Pathology, University of Cambridge, Cambridge, UK

    • Heather Griffin
    •  & John Doorbar
  2. National Institute for Medical Research, London, UK

    • Heather Griffin
    • , Yasmina Soneji
    • , Zhonglin Wu
    •  & John Doorbar
  3. DDL Diagnostic Laboratory, Rijswijk, The Netherlands

    • Romy Van Baars
    • , David Jenkins
    • , Miekel van de Sandt
    •  & Wim Quint
  4. University College Hospital, London, UK

    • Rupali Arora
    •  & Albert Singer
  5. Department of Gynecology and Oncology, Jagiellonian University College, Krakow, Poland

    • Robert Jach
    • , Krzysztof Okon
    •  & Hubert Huras


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Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to John Doorbar.